SCiENTifiC RepoRtS | (2018) 8:4493 | DOI:10.1038/s41598-018-22817-5
Demonstration of the potential of
environmental DNA as a tool for
the detection of avian species
, Koichi Murata
, Tetsuya Sado
, Isao Nishiumi
, Masamichi Takeshita
& Masaki Miya
Birds play unique functional roles in the maintenance of ecosystems, such as pollination and seed
dispersal, and thus monitoring bird species diversity is a rst step towards avoiding undesirable
consequences of anthropogenic impacts on bird communities. In the present study, we hypothesized
that birds, regardless of their main habitats, must have frequent contact with water and that tissues
that contain their DNA that persists in the environment (environmental DNA; eDNA) could be used
to detect the presence of avian species. To this end, we applied a set of universal PCR primers (MiBird,
a modied version of sh/mammal universal primers) for metabarcoding avian eDNA. We conrmed
the versatility of MiBird primers by performing in silico analyses and by amplifying DNAs extracted
from bird tissues. Analyses of water samples from zoo cages of birds with known species composition
suggested that the use of MiBird primers combined with Illumina MiSeq could successfully detect avian
species from water samples. Additionally, analysis of water samples collected from a natural pond
detected ve avian species common to the sampling areas. The present ndings suggest that avian
eDNA metabarcoding would be a complementary detection/identication tool in cases where visual
census of bird species is dicult.
Environmental DNA (eDNA) is genetic material that persists in an environment and is derived from organ-
isms living there, and researchers have recently been using eDNA to detect the presence of macro-organisms,
particularly those living in aquatic/semiaquatic ecosystems
. For example, several sh species inhabiting a
river can be detected by amplifying and sequencing DNA fragments extracted from water samples
methodologies such as quantitative PCR and eDNA metabarcoding. Quantitative PCR requires the design of
species-specic PCR primers and enables quantitative measurements of eDNA of target species
, while eDNA
metabarcoding, which has been becoming a common methodology in eDNA studies, uses a universal primer set
and high-throughput sequencer (e.g., Illumina MiSeq) to enable qualitative detection of eDNA of multiple species
belonging to a target taxon
(but see ref.
Although earlier studies mainly focused on detecting sh/amphibian species (i.e., organisms that have close
associations with water), recent studies have shown that eDNA can be used to detect a diverse group of animals,
. Detecting the presence of animals is possible even if their
habitats are terrestrial
because animals must have, in general, frequent opportunities to contact water in
order to live. e ndings of these recent studies imply that any organism, regardless of its main habitat, can
potentially be detected by using eDNA if we can design suitable primers that enable amplication and identica-
tion of DNA fragments of target organisms and if we can collect appropriate media that contain eDNA.
Wild birds represent an important part of the biodiversity in ecosystems, and they play a unique role in the
maintenance of ecosystem functions. For example, in forest ecosystems, birds can contribute to maintenance of
the tree community by seed dispersal and pollination, and to the reduction of herbivory by predation upon insect
. However, recent increases in anthropogenic impacts on ecosystems, e.g., urbanization and habitat
PRESTO, Japan Science and Technology Agency, Kawaguchi, 332-0012, Japan.
Center for Ecological Research,
Kyoto University, Otsu, 520-2113, Japan.
Yokohama Zoological Gardens ZOORASIA, Kanagawa, 241-0001, Japan.
College of Bioresource Sciences, Nihon University, Kanagawa, 252-0880, Japan.
Natural History Museum and
Institute, Chiba, 260-8682, Japan.
Department of Zoology, National Museum of Nature and Science, Tsukuba,
Ibaraki, 305-0005, Japan.
Department of Biological Sciences, The University of Tokyo, Tokyo, 113-0032, Japan.
Correspondence and requests for materials should be addressed to M.U. (email: email@example.com) or M.M.
Received: 16 October 2017
Accepted: 1 March 2018
Published: xx xx xxxx